The Principal Investigator's long-term objectives are to characterize the biochemical mechanisms that lead to loss of functional viability of insulin secretion from pancreatic beta-cells. Methods are being developed to make dynamic measurements of metabolic flux in islet beta-cells by using multinuclear NMR spectroscopic techniques and stable isotopic tracer methodology. The present proposal seeks to identify which of the several metabolic pathways necessary for coupling beta-cell metabolism with insulin secretion cause the impaired glucose stimulated insulin secretion responsible for maturity-onset diabetes of the young type 3 (MODY-3). This research will augment on-going research of the PI into islet beta-cell physiology. MODY-3 is characterized by the onset of beta-cell dysfunction leading to absence of an appropriate insulin secretory response and severe hyperglycemia. MODY-3 is an inherited defect that has been linked to a dominant negative mutation of the hepatic nuclear factor 1 alpha (HNF-1alpha) transcription factor. Attempts to identify the molecular and functional targets have focused on the HNF-1 alpha knockout mouse model, and a stable insulinoma cell line, INS-1, transfected with the most common mutation associated with MODY-3. These models suggest that impaired glycolytic flux, altered mitochondria! metabolism and oxidative phosphorylation, or upregulation of UCP-2 expression may contribute to the reduction in insulin secretion. The proposed study will provide a comprehensive evaluation of the regulation of mitochondrial oxidative and anaplerotic pathways, complemented by NMR spectroscopic measurements of altered UCP-2 activity and oxidative phosphorytion efficiency, and in these MODY-3 models. It is now appreciated that without significant anaplerotic flux into the Kreb's cycle, mitochondrial ATP production per se is not sufficient for insulin secretion. Quantitative analysis of metabolic pathways, by isotopomer methods, indicates that insulin secretion correlates best with anaplerotic flux through pyruvate carboxylase (PC). The mechanism whereby PC flux couples with insulin secretion may involve transduction of a signal dependent on pyruvate cycling, or may be due to the generation of downstream second messenger. This study will combine isotopomer analysis with siRNA knockdown of malic enzyme (required for pyruvate cycling) to resolve this question and to determine the role that HNF-1 alpha may have on the pathways linking metabolism with insulin secretion.